A longstanding worldwide demand exists for new, effective, environmentally friendly, and safe means to control pests that damage agriculture or serve as disease vectors. Agriculture costs incurred by pests exceed billions of dollars annually in decreased crop yields, reduced crop quality, increased harvesting costs, pesticide application costs, and negative ecological impact. In addition to agriculture pests, many blood-feeding insects are vectors for pathogenic microorganisms that threaten human and animal health, or are annoying at the least. As in the case of agriculture pests, direct and intangible costs incurred by blood-feeding pests concern pesticide safety hazards to humans and animals, bioaccumulation and environmental incompatibility, and synthesis and application costs.
Almost all field crops, nursery and horticulture plants, and commercial farming areas are susceptible to attack by one or more pests. Particularly problematic are Coleopteran and Lepidopteran pests. An example of a Lepidopteran pest is the hornworm larva of Manduca sexta, and an example of a Coleopteran pest is the Colorado potato beetle, Leptinotarsa decemlineata. Vegetable and cole crops, lentils, leafy vegetables, melons, peppers, potatoes and related tubers, tomatoes, cucumbers and related vine crops, as well as a variety of spices are sensitive to infestation by one or more pests including loopers, armyworms, moth larvae, budworms, webworms, earworms, leafeaters, borers, cloverworms, melonworms, leafrollers, various caterpillars, fruitworms, hornworms, and pinworms. Likewise, pasture and hay crops such as alfalfa, pasture and forage grasses and silage are often attacked by a variety of pests including armyworms, alfalfa caterpillars, European skipper, a variety of loopers and webworms, as well as yellowstriped armyworms.
Fruit (including citrus), nut, and vine crops are susceptible to attack by a variety of pests, including sphinx moth larvae, cutworms, skippers, fireworms, leafrollers, cankerworms, fruitworms, girdlers, webworms, leaffolders, skeletonizers, shuckworms, hornworms, loopers, orangeworms, tortrix, twig borers, casebearers, spanworms, budworms, budmoths, and a variety of caterpillars and armyworms.
Field crops are targets for infestation by insects including armyworm, asian and other corn borers, a variety of moth and caterpillar larvae, bollworms, loopers, rootworms, leaf perforators, cloverworms, headworms, cabbageworms, leafrollers, podworms, cutworms, budworms, hornworms, and the like. Pests also frequently feed upon bedding plants, flowers, ornamentals, vegetables, container stock, forests, fruit, ornamental, shrubs and other nursery stock. Even turf grasses are attacked by a variety of pests including armyworms and sod webworms.
For the past 50 years growers, health officials, and the public have depended on chemical pesticides for controlling a variety of pests. However, environmental experts, health officials, and the public have become concerned about the amount of residual chemicals found in food, ground water, and elsewhere in the environment. Regulatory agencies around the world are restricting and/or banning the uses of many synthetic pesticides, particularly those that are persistent in the environment and that enter the food chain. Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides from the market place could limit economical and effective options for controlling costly pests. Some synthetic chemical pesticides can poison the soil and underlying aquifers, pollute surface waters as a result of runoff, and destroy non-target life forms. These synthetic chemical pest control agents have the further disadvantage of presenting public safety hazards when they are applied in areas where pets, farm animals, or children may come into contact with them. They can also pose health hazards to the people applying them, especially if the proper application techniques are not followed.
Because crops of commercial interest are often the targets of pests, environmentally sensitive methods for controlling or eradicating pest infestations are desirable in many instances. This is particularly true for farmers, nurserymen, growers, and commercial and residential areas which seek to control pest populations using environmentally friendly compositions.
The most widely used environmentally friendly pesticidal formulations developed in recent years have been microbial pesticides derived from the bacterium Bacillus thuringiensis (xe2x80x9cB.t.xe2x80x9d). B. thuringiensis is a Gram-positive bacterium that produces proteins which are toxic to certain orders and species of pests. Many different strains of B. thuringiensis have been shown to produce insecticidal proteins. Compositions including B. thuringiensis strains which produce insecticidal proteins have been commercially-available and used as environmentally-acceptable insecticides because they are toxic to specific target pests, but are harmless to plants and other non-target organisms. The specificity of these toxins is often strain-specific, with certain toxins being active against a relatively narrow spectrum of pests. Indeed, many B.t. toxins have been identified that are active only against particular insect orders (e.g., dipterans, hymenopterans, coleopterans, etc.). This limitation prevents the use of a single B.t. endotoxin composition as a broad-range pesticide.
Crop pests are not the only targets for which an environmentally friendly and safe pesticide would be highly desirable. Many blood-feeding pests are known to prey on humans and animals, and many pests are vectors for pathogenic microorganisms that threaten human and animal health, including commercially important livestock, pets and other animals. The order Diptera contains a large number of blood-ingesting and disease-carrying pests, including, for example, mosquitoes, black flies, no-see-ums (punkies), horse flies, deer flies and tsetse flies. Various species of mosquitoes, for example, transmit diseases caused by viruses, and many are vectors for disease-causing nematodes and protozoa. Mosquitoes of the genus Anopheles transmit Plasmodium, the protozoan that causes malaria. The mosquito species Aedes aegypti transmits an arbovirus that causes yellow fever in humans. Other viruses transmitted by Aedes species include the causative agents of dengue fever, eastern and western encephalitis, Venezuelan equine encephalitis, St. Louis encephalitis, chikungunya, oroponehe and bunyarnidera The genus Culex, which includes the common house mosquito C. pipiens, is implicated in the transmission of various forms of encephalitis, filarial worms, and West Nile virus. Trypanasoma cruzi, the causative agent of Chagas disease, is transmitted by various species of blood ingesting Triatominae bugs. Tsetse flies (Glossina spp.) transmit African trypanosomal diseases of humans and cattle. Other diseases are transmitted by various blood-ingesting pest species.
Various pesticides have been employed in efforts to control or eradicate populations of disease-transmitting pests. For example, DDT, a chlorinated hydrocarbon, has been used in attempts to eradicate malaria-bearing mosquitoes throughout the world. Other examples of chlorinated hydrocarbons are BHC, lindane, chlorobenzilate, methoxychlor, and the cyclodienes (e.g., aldrin, dieldrin, chlordane, heptachlor, and endrin). The long-term stability of many of these pesticides and their tendency to bioaccumulate render them particularly dangerous to many non-target organisms.
In addition to environmental concerns, another major problem associated with conventional chemical control practices is the capability of many species to develop pesticide resistance. Resistance results from the selection of naturally occurring mutants possessing biochemical, physiological or behavioral factors that enable the pests to tolerate the pesticide when it is applied.
There is clearly a longstanding need in the art for pesticidal compounds that reduce or eliminate direct and/or indirect threats to human health posed by presently available pesticides, that are environmentally compatible and safe, are not toxic to non-pest organisms, and have a reduced tendency to bioaccumulate.
Approaches to pesticide development arc lacking that involve specifically disrupting key pest metabolic regulatory processes, notably membrane transporter or channel proteins as targets. The development of such methodologies could provide safer, environmentally friendly alternatives to conventional commercially used pesticides, and provide more economical means for suppressing or eradicating target pest populations. The formulation of pesticidal compositions that are non-toxic to animals and to humans would greatly enhance the present methods available for killing pests, and would provide alternative strategies for environmentally responsible pest management.
Membrane transporter proteins and ion channel proteins serve critical roles in maintaining organic solute and ionic metabolic, thermodynamic, and electrical events in all cells. In both eukaryotes and prokaryotes these proteins affect electrochemical gradients of a wide variety of metabolic molecules and electrolytes, including amino acids and related metabolites as well as H+, OHxe2x88x92, Na+, K+, Clxe2x88x92, and carbonate ions (Gerencser and Stevens, 1994, J. Exper. Biol. 196:59-75; Stevens, B. R. 2001. xe2x80x9cTheory and methods in nutrient membrane transport.xe2x80x9d In: Surgical Research. pp. 845-856. W. W. Souba and D. W. Wilmore, eds., Academic Press, San Diego). Molecular cloning studies have identified several subfamilies of organic solute transporters and ion channels (Griffith, J. K. and C. E. Sansom, 1998, In: The Transporter Facts Book, Academic Press, San Diego, pp. 500).
Organic solute transporters and ion channels are commonly defined by their substrate selectivity within polypeptide superfamilies. For cloned or native secondary active transporters, it is generally assumed that cell membranes utilize ion and organic molecule electrochemical gradients to aid in exchanging these solutes between the cell interior and extracellular environment (Gerencser, G. A. and B. R. Stevens, 1994, J. Exper. Biol. 196:59-75; Stevens, B. R. 1999, Digestion and Absorption of Protein. In: Biochemical and Physiological Aspects of Human Nutrition. pp. 107-123, M. H. Stipanuk, ed., W. B. Saunders Co., Philadelphia). In the xe2x80x98prototypicalxe2x80x99 transporter, organic solutes that can be moved across cell membranes by uniport, hetero- or homo-exchange, and/or uptake can be activated by ions, and/or thermodynamiclly cotransported with ions (Quick, M. and B. R. Stevens, 2001, J. Biol. Chem. 276(36):33413-33418; Griffith, J. K. and C. E. Sansom, 1998, In: The Transporter Facts Book, Academic Press, San Diego, pp. 500). Ion channels, on the other hand, are typically distinct from organic solute transporters, are selective in their conducting ion species, and may be gated by organic ligands (Hille, B, 2001, Ionic channels of excitable membranes, 3rd Edition, Sinauer Associates, Inc., Sunderland, Mass., pp. 814).
Manduca sexta is a major crop pest whose larval stage, commonly known as tobacco and tomato hornworms, rapidly attack and defoliate tobacco and tomato plants; the large fifth instar larvae are especially damaging. Other vegetable crops such as peppers, eggplant, and potatoes also can be affected. Tobacco and tomato hornworms rapidly grow and gain weight as they progress from the first instar stage (about 6.7 mm) through the fifth instar (about 81.3 mm) over a period of about 20 days. The sated larvae then drop to the soil to pupate, and eventually emerge as adult moths. The moths lay eggs, which develop into larvae, and the life cycle continues, thereby sustaining crop damage. Killing the larvae prevents immediate crop damage and prevents or reduces future damage by interrupting the life cycle.
The midgut region of M. sexta larvae displays compartments with the property of high concentrations of K+ as well as Na+ in an alkaline fluid (xcx9cpH 10), with trans-epithelial potentials xcx9c250 mV (Harvey et al., 1999, Am. Zool. 38:426-441; Harvey and Wieczorek, 1997, J. Exper. Biol. 200:203-216). Epithelial cells of this region transport a variety of nutrients, including nutrient amino acids and electrolytes, as demonstrated by in vitro isolated membrane vesicle uptake studies. In place of a Na+/K+-ATPase typically found in cells, this tissue instead possesses a proton translocating V-ATPase (Graf et al., 1992, FEBS Lett. 300:119-122; Merzendorfer et al., 1997, J. Exper. Biol. 200:225-235) which energizes the cell membranes for secretion and absorption of K+ and Na+ ions, and establishment of a large pH gradient. A K+-activated leucine-preferring transporter (KAAT1) has been identified from the hornworm midgut (Castagna et al., 1998, Proc Natl. Acad Sci. USA 95:5395-5400), and a GABA (gamma aminobutyric acid) transporter has been cloned from an M. sexta embryo cDNA library (Mbungu et al., 1995, Arch. Biochem. Biophys. 318:489-497).
CAATCH1 (Cation-Amino Acid Transporter/CHannel) is a recently cloned insect membrane protein initially cloned from Manduca sexta; CAATCH1 exhibiting a unique polypeptide and nucleotide sequence related to, but different from, mammalian Na+, Clxe2x88x92-coupled neurotransmitter transporters (Feldman et al., 2000, J. Biol. Chem. 275:24518-24526). Utilizing a unique PCR-based strategy, the gene encoding CAATCH1 was cloned (Feldman et al., 2000, supra) from a cDNA library in LambdaZap plasmids, obtained from the digestive midgut of Manduca sexta larvae.
The unanticipated and novel biochemical, physiological, and molecular properties of CAATCH1 indicated that it is a multi-function protein that mediates amino acid uptake in a manner that is thermodynamically uncoupled from ion electrochemical potentials, and furthermore that CAATCH1 simultaneously functions as an amino acid-modulated gated alkali cation channel (Quick, M., and B. R. Stevens, 2001, xe2x80x9cAmino acid transporter CAATCH1 is also an amino acid-gated cation channelxe2x80x9d. J. Biol. Chem 276: 33413-33418) serving at least Na+ and K+. Radiotracer and electrophysiology experiments with functional CAATCH1 polypeptide expressed from the full length CAATCH1 cDNA demonstrated direct amino acid ligand-protein interactions, and indicated that binding by different amino acid substrates differentially affects the conformational states of CAATCH1 (Quick, M. and B. R. Stevens, 2001, J. Biol. Chem. 276:33413-33418). Notably, L-methionine binding to CAATCH1 in situ in biomembranes in the presence of Na+ perturbs the charge-voltage relation with a high affinity binding constant, affecting transient currents due to CAATCH1-associated charge transfer across the membrane dielectric field. Furthermore, CAATCH1-associated voltage-dependent amino acid-elicited steady state inward cation currents are blocked by methionine, and indeed methionine reversed charge movements via CAATCH1 expressed in cell membranes, even though radiotracer methionine influx is catalyzed by CAATCH1 (Quick, M. and B. R. Stevens, 2001, J. Biol. Chem. 276:33413-33418). In insects, CAATCH1 likely plays a broader role than that of a xe2x80x98classicalxe2x80x99 transporter or channel; as a multifunction protein CAATCH1 is likely a key protein in electrolyte and organic solute homeostasis of certain insects (Feldman et al, 2000, J. Biol. Chem. 275(32):24518-24526)
Many pestsxe2x80x94including mosquitoes, Lepidopterans, and Coleopteransxe2x80x94possess an alkaline pH midgut, and share some similar physiological mechanisms that occur within this unusual milieu (Nation, J., 2001, In: Insect Physiology and Biochemistry, CRC Press, Boca Raton, pp 496). Mosquito larvae possess such an alkaline midgut, and adjust free amino acid concentrations in their hemolymph and extracellular compartments in direct response to existence of foreign factors in the gut (e.g., B.t. xcex4-endotoxin) or the salinity of their habitat (Bounias, M. et al., 1989, J. Invertebr Pathol. 54:16-22). Notably, one standout free amino acid, L-proline, accumulates 4-fold during the normal course of Aedes aegypti larval development, and in Culex spp. L-proline accumulation can exceed 50-fold (up to 70 mM) when larvae are stimulated by Na+ in their feeding pools (Bounias, M. et al., 1989, supra; Patrick, M. L. and T. J. Bradley, 2000, J. Exp. Biol. 203:831-839; Chaput, R. L. and J. N. Liles, 1969, Ann. Entomol. Soc. Am. 62:742-747). This proline is likely utilized as an energy source and for osmoregulation (Patrick, M. L. and T. J. Bradley, 2000, J. Exp. Biol. 203:831-839; Bounias et al., 1989, J. Invertebr Pathol. 54:16-22). In contrast, free L-methionine has the distinction of virtually the lowest measurable concentration ( less than 0.001 mM) of any of the free amino acids in mosquito larvae. Virtually all methionine existing in the free amino acid state in larvae (Dadd, R. H., 1973, Ann Rev Entomol 18:381-420) is metabolically shunted and sequestered into so-called methionine-rich hexamerin proteins (Korochkina et al., 1997, Insect Biochem Mol Biol. 27:813-824) that are stored for post-larval developmental events. In its role as a nutrient transporter, the CAATCH1 protein has been shown to be primarily responsible for proline uptake (Feldman et al., 2000, J. Biol. Chem. 275(32):24518-24526), while in the presence of extremely low concentrations of methionine, CAATCH1 effectively shuts down ionic fluxes via its channel properties (Feldman et al., 2000, J. Biol. Chem. 275(32):24518-24526; Quick, M. and B. R. Stevens, 2001, J. Biol. Chem. 276(36):33413-33418).
Compared to conventional organic pesticides, the use of biodegradable small molecules, such as amino acids, as pesticides is highly desirable, owing to the safety of such compounds to humans, animals, and the environment.
The present invention provides materials and methods for pest control. In a preferred embodiment, the subject invention overcomes drawbacks inherent in the prior art by providing pesticidal compositions that contain one or more compounds that interact with organic solute transporter/ligand-gated ion channel multifunction polypeptides in the pest. Upon exposure, ingestion, or other means of absorption by a target pest, these compositions either compromise pest growth and/or cause the death of the pest. In a preferred embodiment, the compositions of the subject invention contain one or more amino acids and/or amino acid analogs.
In a preferred embodiment, the materials and methods of the subject invention achieve pest control by disrupting the function of a newly discovered class of multifunction solute transporter/ligand-gated ion channel proteins (the CAATCH1 class of proteins). The CAATCH1 protein of Manduca sexta (tomato hornworm) exemplifies this class of proteins. In accordance with the subject invention, CAATCH1 has been found to be a key protein in regulating electrolyte and organic solute fluxes, especially in those pests with midguts exhibiting an alkaline pH. The unanticipated biochemical, physiological, and molecular properties of CAATCH1 indicated that it is a multi-function protein that mediates amino acid uptake in a manner that is thermodynamically uncoupled to ion electrochemical potentials, and furthermore that CAATCH1 simultaneously functions as an amino acid-modulated gated alkali cation channel serving at least Na+ and K+. In accordance with the subject invention, effective pest control is achieved by disrupting the function of a CAATCH1 protein and/or related molecules.
As described herein, the function of the CAATCH1 proteins, and related proteins, can be disrupted in a number of ways to achieve the desired pest control. For example, in accordance with the subject invention, it has been found that small molecules, which interfere with these proteins, can be administered to a pest, or its situs, to achieve pest control. Specifically exemplified herein is the use of the amino acids methionine or leucine (and/or analogs thereof) to control pests. These compounds can be administered in a wide range of ways including the application of compositions comprising these individual amino acids (and/or their analogs, or bound to other molecules, for example by amide bonds), application of polypeptides which are made up of an abundance of these pesticidal amino acids, and providing a transgenic plant which expresses a pesticidal amount of the amino acids as either free amino acids, as salts of amino acids or their analogs, or bound to other molecules, or existing in polypeptides.
In a preferred embodiment, the methods of the subject invention involve delivering to or applying to a pest a composition that comprises methionine or leucine, or an analog thereof. Exemplary analogs include, but are not limited to, methionine esters, leucine esters, D-methionine, D-leucine, D-tert-leucine, L-tert-leucine, DL-methionine, DL-leucine, L-methioninol, L-leucininol, L- or D-methioninemethylsulfonaium chloride, small methionyl peptides, low molecular weight leucyl peptides, alphaketoisocaproic acid, and the like. Likewise, methyl or ethyl esters of such compounds are also contemplated to be useful, as are keto derivatives of such compounds. In a specific embodiment, insect pests are controlled by administering methionine to the insects. Alternatively, other amino acids such as histidine, glycine, threonine, or alanine can also be used. For all embodiments involving compounds with chiral centers, the L-, D-, DL-, or partially racemic forms are also contemplated to be useful. Alternatively, amino acid analogs that do not possess chiral centers are contemplated to be useful.
Additional target crops to be protected within the scope of the present invention comprise, e.g., the following species of plants:
Although it is believed that the administration of amino acids to achieve pest control according to the subject invention is effective as a result of the disruption of particular proteins as described herein, one aspect of the subject invention is simply the control of pests by administering amino acids or these analogs (regardless of the specific mechanism involved).
One aspect of this invention contemplates altering methionine levels in pests by manipulating biochemical pathways leading to methionine production in target pests. Such manipulations include, but are not limited to, precursors and cofactors of methionine metabolic biosynthesis pathways.
The subject invention provides various other alternative approaches to disrupting the function of the unique newly discovered class of multifunction transporter/channel proteins. These other approaches include, for example, the use of interfering RNA (RNAi), gene silencing techniques, and antisense polynucleotides. In a specific embodiment, the antisense molecules are complementary to contiguous nucleotide sequences of at least about 15 nucleotides from SEQ ID NO:1. These antisense constructs can be used to xe2x80x9cdown-regulatexe2x80x9d the expression of CAATCH1, and/or related proteins, in a particular cell. Alternatively, antisense constructs complementary to a contiguous nucleotide sequence from the CAATCH1 promoter sequence may also be used to regulate the activity of CAATCH1 and/or related proteins. Antisense constructs are well-known in the art and include the use of antisense mRNA to reduce the transcription or translation or otherwise impair the net production of the encoded polypeptide.
The subject invention also provides methods for identifying compounds which regulate, alter, or modulate the biochemical and/or physiological functional activity of a CAATCH1 polypeptide or polynucleotide (or related molecules). In one embodiment this method comprises exposing a cell that expresses a CAATCH1 polypeptide to at least one compound or signal whose ability to modulate the activity of the CAATCH1 polypeptide is sought to be determined, and thereafter monitoring the cell for a change that is a result of the modulation of activity of CAATCH1 or related polypeptide(s). Such an assay is particularly contemplated to be useful in the identification of agonists, antagonists and/or allosteric modulators of CAATCH1.
A further aspect of the invention provides methods for screening compounds (e.g., synthetic peptides, peptide analogs, peptidomimetics, small molecule inhibitors, etc.) which inhibit or reduce the binding of a CAATCH1 polypeptide. According to this embodiment, screening for chemical or biochemical entities may be performed e.g., by means of a cell-based assay, an in vitro assay for CAATCH1function and/or rational pesticidal formulation or amino acid transporter-active analogs, drugs, or compounds. Cell-based assays for screening can be designed e.g., by constructing cell lines in which the expression of a reporter protein, i.e. an easily assayable protein, is dependent on CAATCH1 activity. Such an assay enables the detection of compounds that directly antagonize CAATCH1, or compounds that inhibit other cellular functions required for the activity of CAATCH1. Compounds may also be identified which recognize or inhibit amino acid or other solute transport via the CAATCH1 polypeptide or modify ion flux through the CAATCH1 polypeptide. Example ions include, but are not limited to, N+, K+, H+, OHxe2x88x92, Clxe2x88x92, bicarbonate, and carbonate.
In another aspect, the present invention provides an antibody that is immunoreactive with a transporter/channel polypeptide of the invention. Reference to antibodies includes whole polyclonal and monoclonal antibodies, and parts thereof, either alone or conjugated with other moieties. The monoclonal antibodies of the present invention can be used in standard immunochemical procedures, such as immunoprecipitation, ELISA and Western blot methods. Also, immunoabsorbent protocols may be used in purifying native or recombinant peptide species or synthetic or natural variants thereof.
Advantageously, the amino acid-based targeted pesticides of the subject invention are environmentally safe due to target selectivity, low toxicity to humans and pets, and their biodegradation by environmentally friendly naturally occurring microorganisms. Also, the use of these pesticides is compatible with the use of natural enemies of pests (e.g., parasitoids and predators). In fact, L-methionine is an xe2x80x9cessential or indispensablexe2x80x9d amino acid in humans, meaning that L-methionine is not synthesized by the body but instead is a required nutrient in the diet needed to sustain human life (Fuller, M. F., 2000. xe2x80x9cProtein and amino acid requirementsxe2x80x9d, pp.287-304 In: Biochemical and Physiological Aspects of Human Nutrition, M. H. Stipanuk, ed. W. B. Saunders Co., Philadelphia). Since it is a naturally occurring substance, the use of this compound and related amino acids contribute to a sustainable, pesticide-free food supply, and preserve the environment by reducing the reliance on traditional pesticides.